CN108901844B - Method for constructing lycoris genus genetic transformation system - Google Patents
Method for constructing lycoris genus genetic transformation system Download PDFInfo
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Abstract
The invention relates to a construction method of a lycoris genus genetic transformation system, belonging to the technical field of genetics. A method for constructing the genetic transformation system of Lycoris includes such steps as introducing the target exogenous gene to the callus of Lycoris by genetic engineering, differential culture and strong seedling culture to obtain the test-tube plantlet of Lycoris, and features that the target exogenous gene is used to control the biological characteristics of Lycoris. The construction method provided by the invention has the characteristics of good repeatability and high success rate, and the success rate reaches 60-80%. The establishment of the lycoris genetic transformation system opens up a new way for breeding new varieties of lycoris.
Description
Technical Field
The invention belongs to the technical field of genetics, and particularly relates to a method for constructing a lycoris genus genetic transformation system.
Background
Lycoris radiata (Lycoris Herb.) belongs to Amaryllidaceae (Amaryllidaceae), and is a perennial Herb. The bulbs are in an egg-shaped shape, leaves come out in spring, leaves are in a belt shape, and 4-6 flowers are arranged in an umbrella-shaped inflorescence; yellow, red, white; the quilt flap strength rolls back and collapses. The flowering period is 7-8 months. The flower stem is tall and straight, the flower shape is beautiful, and the flower color is gorgeous. The flower has high ornamental value in all kinds, and is gradually novel fresh cut flowers, garden ground covers and potted bulbous flowers.
However, at present, the use of lycoris plants in China is limited to domestication and cultivation of wild resources. The breeding and popularization of new variety of lycoris is only in the initial stage. At present, only plant research institutes of China academy of sciences of Jiangsu province obtain 1 each provincial new variety through crossbreeding and systematic breeding; and 4 new species are internationally registered in the Hangzhou vegetable garden through systematic breeding. Because part of the species of the genus belongs to the hybrid origin and the fertility is poor, the new variety is difficult to obtain through hybrid breeding; even if the hybridization was successful, the rate of the hybridization was poor and the positive rate was low.
Disclosure of Invention
In view of the above, the invention aims to provide a method for constructing a lycoris genetic transformation system, and opens up a new way for breeding new varieties of lycoris.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for constructing a lycoris genetic transformation system, which comprises the following steps:
(1) carrying out enzyme digestion, connection and screening on the exogenous gene and the vector in sequence to obtain a recombinant vector;
(2) introducing the recombinant vector into agrobacterium-mediated cells, carrying out shake culture for 2-3 h, centrifuging, collecting thalli, coating the thalli on a flat plate containing 50-150 mg/L kanamycin resistance, and screening single colonies to obtain positive recombinant agrobacterium;
(3) transferring the positive recombinant agrobacterium to an LB liquid culture medium, culturing at 25-29 ℃ and 180-220 rpm for more than 16h by shaking table until the OD of the bacterial liquid600Obtaining a positive recombinant agrobacterium liquid when the value reaches 0.4-0.6;
(4) infecting the lycoris callus with the positive recombinant agrobacterium liquid for 25-35 min to obtain infected lycoris callus;
(5) inoculating the infected lycoris callus on an MS culture medium, and carrying out dark culture for 2-4 d at 24-26 ℃ to obtain dark-cultured lycoris callus;
(6) inoculating the dark cultured lycoris callus on a flat plate containing antibiotics to perform callus differential culture; subculturing every 19-22 days until callus is differentiated to obtain cluster buds;
(7) inoculating the cluster buds to a bud-strengthening culture medium containing antibiotics for bud-strengthening culture, and subculturing once every 19-22 days until bulblets are obtained;
(8) and (4) inoculating the bulblets on a rooting culture medium again for rooting culture to obtain test-tube plantlets with 3-5 roots.
Preferably, the antibiotic in the antibiotic-containing plate in step (6) comprises the following components in concentration: 200-300 mg/L kanamycin and 150-250 mg/L timentin;
the culture medium in the plate is MS culture medium containing the following components in percentage by weight: 30g/L of sucrose, 7.5g/L of agar, 6.0mg/L of 6-BA, 1.5mg/LNAA and 1.0mg/L of multi-wall carbon nano-tube; the pH value of the culture medium is 5.6-5.8.
Preferably, the temperature of dark culture in the step 5) is 24-26 ℃.
Preferably, the light intensity of the subculture in the step 6) is 100 to 300 [ mu ] mol/m-2·s-1The illumination time is 16 h.
Preferably, the budding medium in the step (7) comprises an MS medium with the following components: sucrose 40g/L, agar 7.5g/L, 2.0mg/L6-BA and 0.5 mg/LNAA; the pH value of the strong bud culture medium is 5.6-5.8.
Preferably, the conditions of the bud-strengthening culture in the step (7) and the co-culture in the step (8) are as follows: the culture temperature is 24-26 ℃, and the illumination intensity is 100-300 mu mol.m-2·s-1And the illumination time is 16 h.
Preferably, the preparation method of the lycoris callus in the step (4) comprises the following steps:
s1, transferring the bulblets of the Lycoris to an induction culture medium, and performing dark culture at 24-26 ℃ for 28-32 days to obtain a light yellow callus mass;
s2, carrying out subculture on the faint yellow callus masses until the diameter of the induced callus masses exceeds 1cm, and finishing the culture.
Preferably, the induction medium comprises MS medium at the following levels: sucrose 30g/L, agar 7.5g/L, 6.0mg/L6-BA and 1.5 mg/LNAA.
Preferably, the exogenous gene in the step (1) comprises an anthocyanin synthesis related gene, a galantamine synthesis related gene or a GUS gene.
Preferably, the vector for transferring the GUS gene in the step (1) is pEF-3-GUS-2; the carrier used for transferring the anthocyanin to synthesize the related gene is pCAMBIA 2301; the multiple cloning sites of the insertion vector of the transferred anthocyanin synthesis related gene are SalI and XbaI.
The invention provides a method for constructing an amaryllidachia genetic transformation system, which is characterized in that target exogenous genes are introduced into amaryllidachia callus by means of genetic engineering, and then are subjected to differentiation culture and strong seedling culture to obtain amaryllidachia test-tube plantlets, so as to obtain new amaryllidachia varieties with target exogenous gene controlled biological characters. The construction method provided by the invention has the characteristics of good repeatability and high success rate, and the success rate reaches 60-100%. The establishment of the lycoris genetic transformation system opens up a new way for breeding new varieties of lycoris.
Furthermore, the construction method provided by the invention further limits the induction method of the callus, and because the tissue culture technology of the lycoris in the prior art is lagged, the successful induction culture of the lycoris callus overcomes the technical obstacles in the prior art, and provides a technical basis for the tissue culture of the lycoris. The construction method provided by the invention uses the lycoris callus as the starting material to construct the genetic transformation system, so that the transformation power is improved, and a stable standard operation flow is provided for the construction of the lycoris genetic transformation system.
Furthermore, the construction method provided by the invention further limits the types of exogenous genes, and various alkaloids (such as galanthamine) contained in the lycoris bulb are effective active ingredients for clinically treating senile dementia, but the content is extremely low, and through the establishment of the lycoris genetic transformation system, one or more genes in a synthesis way of adding the galanthamine are transferred to improve the content of the galanthamine; when the exogenous gene is related to anthocyanin synthesis, the flower color or leaf color of the lycoris can be improved. The construction method provided by the invention is beneficial to enriching the lycoris strain and provides great ornamental and medicinal values for novel fresh cut flowers, garden ground covers and potted bulbous flowers.
Drawings
FIG. 1 is a schematic diagram showing the insertion positions of a GUS gene and a MYB119 gene in a vector in the present invention.
Detailed Description
The invention provides a construction method of an Lycoris genetic transformation system, which comprises the following steps:
(1) carrying out enzyme digestion, connection and screening on the exogenous gene and the vector in sequence to obtain a recombinant vector;
(2) introducing the recombinant vector into agrobacterium-mediated cells, carrying out shake culture for 2-3 h, centrifuging, collecting thalli, coating the thalli on a flat plate containing 50-150 mg/L kanamycin resistance, and screening single colonies to obtain positive recombinant agrobacterium;
(3) transferring the positive recombinant agrobacterium to an LB liquid culture medium, carrying out shaking table expansion at the temperature of 25-29 ℃ and the rpm of 180-220 for more than 16h until the OD of the bacterial liquid600The value reaches 0.4-0.6 to obtain a positive recombinant agrobacterium liquid (4), and the positive recombinant agrobacterium liquid is used for infecting the lycoris callus for 25-35 min to obtain infected lycoris callus;
(5) inoculating the infected lycoris callus on an MS culture medium, and carrying out dark culture for 2.5-4 d at the temperature of 24-26 ℃ to obtain dark-cultured lycoris callus;
(6) inoculating the dark-cultured lycoris callus onto a flat plate containing antibiotics for differential culture, and carrying out subculture every 19-22 days to obtain cluster buds;
(7) inoculating the cluster buds to a bud-strengthening culture medium for bud-strengthening culture for 30-35 days, and subculturing once every 19-22 days until bulblets are obtained;
(8) and (4) inoculating the bulblets on a rooting culture medium again for rooting culture to obtain test-tube plantlets.
The invention obtains the recombinant vector by enzyme digestion, connection and screening of the exogenous gene and the vector in sequence. In the present invention, the foreign gene preferably includes an anthocyanin synthesis-related gene or a galantamine synthesis-related gene or a GUS gene.
In the present invention, the anthocyanin synthesis-associated gene is preferably MYB 119. The source of MYB119 is preferably synthesized by a gene synthesis company. While MYB119 is synthesized, enzyme cutting sites SalI and XbaI are added at two ends of a nucleotide sequence of the MYB119, wherein the nucleotide sequence of the MYB119 is shown as SEQ ID No.3 (containing the enzyme cutting sites) in a sequence table.
In the invention, the anthocyanin synthesis gene and the vector are subjected to double enzyme digestion respectively. In the examples of the present invention, the vector was pCAMBIA2301 (with GUS reporter gene).
When a genetic transformation system is constructed by taking the anthocyanin synthesis gene as an exogenous gene, the GUS gene is preferably used as a reporter gene and is simultaneously used in vector construction with the anthocyanin synthesis gene to obtain a recombinant vector. The insertion sites of the GUS gene and MYB119 on the vector are shown in FIG. 1.
After enzyme digestion, the anthocyanin synthesis gene enzyme digestion product and the carrier enzyme digestion product are connected to obtain a connection product. The enzyme for ligation is preferably T4 ligase. The conditions for the attachment are as follows: the reaction was carried out at 25 ℃ for 15 min. The volume ratio of the enzyme digestion product of the GUS gene to the carrier enzyme digestion product is 3: 1. The concentration of the enzyme cutting product of the anthocyanin synthetic gene is preferably 20 ng/L.
After obtaining the ligation product, the invention screens the ligation product. The screening method comprises the following steps: and introducing the ligation product into agrobacterium, coating the obtained agrobacterium on a flat plate containing antibiotics for culture, selecting a white single colony, carrying out amplification culture, and carrying out positive cloning detection to obtain the recombinant vector.
In the present invention, when GUS gene is used as a foreign gene, a specific method for constructing a genetic transformation system is described. The recombinant vector transferred with GUS gene is pEF-3-GUS-2. The pEF-3-GUS-2 was purchased from Addgene.
In the present invention, the introduction method is preferably a method of heat shock at 42 ℃ for 30 s. The agrobacterium is preferably an agrobacterium strain EHA105 competent cell. The Agrobacterium introduced with the ligation product was cultured for 1h on antibiotic-free plates with adaptive shaking. The adaptive shaking culture is beneficial to restoring the activity of the agrobacterium introduced with the ligation product. The antibiotic in the antibiotic-containing plate is preferably 50mg/L Km. The culture temperature is preferably 37 ℃, and the culture time is preferably 8-12 h.
The method for detecting the positive clone preferably comprises colony PCR detection of the positive clone and sequencing verification. The sequence after sequencing is consistent with the sequence of GUS gene, which indicates that the ligation product is a recombinant vector.
After obtaining the recombinant vector, the recombinant vector is introduced into agrobacterium-mediated cells, shake culture is carried out for 2-3 h, centrifugation is carried out, thalli are collected, the thalli are coated on a flat plate containing 50-150 mg/L kanamycin resistance, and single bacterial colony is screened, so that the positive recombinant agrobacterium is obtained.
In the present invention, the ratio of the volume of the competent cell solution to the mass of the recombinant vector is 100 μ L: 0.01 to 1.0. mu.g. The competent cell agrobacterium strain EHA105 competent cell. The source of the competent cells of said Agrobacterium strain EHA105 is not particularly limited in the present invention, and commercially available routes well known in the art are used. The temperature for the shaking culture is preferably 28 ℃. The rotation speed of the centrifugation is preferably 6000 rpm. The time for the centrifugation is preferably 1 min. The culture medium of the plate is LB culture medium. And screening the single colony, namely detecting positive clones by colony PCR (polymerase chain reaction), and sending to sequencing verification. The specific method is the same as the screening process of the scheme.
After the positive recombinant agrobacterium is obtained, the invention infects the lycoris callus for 30-40 min by the positive recombinant agrobacterium to obtain the infected lycoris callus.
In the invention, the preparation method of the lycoris callus comprises the following steps:
s1, transferring the bulblets of the Lycoris to an induction culture medium, and performing dark culture at 24-26 ℃ for 28-32 days to obtain a light yellow callus mass;
s2, carrying out subculture on the faint yellow callus masses until callus masses with the diameter more than 1cm are induced.
In the present invention, the induction medium preferably includes MS medium in the following contents: sucrose 30g/L, agar 7.5g/L, 6.0mg/L6-BA and 1.5 mg/LNAA. The temperature of the dark culture is preferably 25 ℃. The dark culture time is preferably 30 days. The condition of the subculture is the same as that of dark culture. The culture medium for subculture is the same as the induction medium.
In the present invention, the infestation will preferably be about 1cm in volume3The lycoris radiata callus is put into the MS liquid culture medium containing the transformed agrobacterium and continuously shaken, which is beneficial to the transformed agrobacterium to invade the callus.
After the infected lycoris callus is obtained, the infected lycoris callus is inoculated on an MS culture medium and is subjected to dark culture for 3 days at the temperature of 24-26 ℃ to obtain the dark-cultured lycoris callus. The dark culture is beneficial to restoring the vitality of the lycoris callus in a short time. The temperature of the dark culture is preferably 25 ℃.
After the lycoris callus is cultured in the dark, the lycoris callus cultured in the dark is inoculated on a flat plate containing antibiotics for dark culture, and subculture is carried out once every 20 days to obtain cluster buds.
In the present invention, the antibiotic in the antibiotic-containing plate preferably includes the following components in concentrations: 200-300 mg/L kanamycin and timentin 150-250 mg/L, more preferably 250mg/L kanamycin and timentin 200 mg/L. The culture medium in the plate is MS culture medium containing the following components in percentage by weight: 30g/L of sucrose, 7.5g/L of agar, 6.0mg/L of 6-BA, 1.5mg/LNAA and 1.0mg/L of multi-wall carbon nano-tube; the pH value of the culture medium is 5.6-5.8.
In the invention, the temperature of the differentiation culture is preferably 24-26 ℃, and more preferably 25 ℃. The illumination intensity of the subculture is preferably 100-300 [ mu ] mol · m-2·s-1The illumination time is 16 h. The temperature of the subculture is the same as the conditions of the differentiation culture.
After cluster buds are obtained, the cluster buds are inoculated on a bud-strengthening culture medium for bud-strengthening culture for 30-35 days, and subculture is carried out once every 19-22 days until bulblets are obtained.
In the present invention, the strong bud medium is preferably an MS medium comprising the following components in contents: sucrose 40g/L, agar 7.5g/L, 2.0mg/L6-BA and 0.5 mg/LNAA; the pH value of the strong bud culture medium is 5.6-5.8.
In the invention, the temperature for culturing the strong bud is preferably 24-26 ℃, and more preferably 25 ℃. The time for culturing the strong seedlings is preferably 32-34 d. The strong bud culture is preferably performed under light conditions. The illumination intensity is 100 to 300 [ mu ] mol/m-2·s-1More preferably 150 to 250. mu. mol/m-2·s-1. The illumination time is 16 h.
After the bulblets are obtained, the bulblets are inoculated on a rooting culture medium for rooting culture to obtain test-tube plantlets. The rooting culture medium comprises an MS culture medium with the following components in percentage by weight: 40g/L of sucrose, 7.5g/L of agar and 1.5g/L of multi-wall carbon nano-tube. The rooting culture temperature is 24-26 ℃, and more preferably 25 ℃. The illumination intensity is 100 to 300 [ mu ] mol/m-2·s-1More preferably 200. mu. mol. m-2·s-1. The illumination time is 16 h. After co-cultivation for 30 days, the test-tube plantlets can grow into 3-5 root-system plantlets with the diameter of 1-2 mm.
After obtaining the test-tube plantlet, the invention preferably identifies the transgenic plant of the test-tube plantlet. The identification comprises staining identification and sequencing verification. The leaf of the test-tube plantlet is cut by 0.3cm for dyeing identification, and the test-tube plantlet is placed in GUS dye solution and dyed for more than 12 hours at 37 ℃. Then, absolute ethyl alcohol is used for decoloring. And if the leaves are blue, the reporter gene is successfully integrated into the plant genome, and the plant is a transgenic plant. When the technology is adopted for lycoris transgenic, the dyeing rate of positive plants is 60-65%. The sequencing validation included PCR amplification of all blue-stained plants. The primer for PCR amplification comprises a forward primer and a reverse primer; the nucleotide sequence of the forward primer is as follows: 5'-GCTGGTCACCAATTCACACG-3' (SEQ ID No. 1); the nucleotide sequence of the reaction primer is as follows: 5'-AGAACACGGGGGACTCTTGA-3' (SEQ ID No. 2). The blue-dyed plants can amplify GUS genes, and the amplification rate is 100%.
The method for constructing a genetic transformation system of Lycoris provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Establishment of lycoris GUS transgenic system
1 induction of callus the aseptic bulblets of Lycoris chinensis were transferred to sucrose 30g/L + agar 7.5g/L + MS +6.0mg/L6-BA +1.5mg/LNAA medium. After 2 weeks, white protrusions, which are the initiation of callus, appeared around the bulblets, pale yellow tissue appeared around the white protrusions after 20 days, and pale yellow callus clumps appeared after 30 days, at which time subculture was performed again until callus clumps with a diameter of more than 1cm were induced. Culturing at 25 + -1 deg.C in dark.
2 construction of the vector
(1) The cloning vector EF-3-GUS-21. mu.L is taken as a recombinant plasmid and is carried into agrobacterium.
(2) And (3) transformation: taking an agrobacterium strain EHA105 stored at the temperature of 70 ℃ below zero, melting on ice, adding 4 mu L of a connecting product when the strain is in a semi-melting state, flicking and uniformly mixing, carrying out ice bath for 20-30min, carrying out heat shock for 30s at the temperature of 42 ℃, immediately placing on ice for 2min, adding 250 mu L of a nonresistant LB liquid culture medium into a super clean workbench, and carrying out shake culture at the temperature of 37 ℃ for 1 hour at 200 rpm.
(3) Coating a plate: after centrifugation, the supernatant was removed, 100. mu.L of the bacterial solution was spread evenly on a 50mg/LKm resistant plate and cultured at 37 ℃ for 8-12 hours.
(4) And (3) positive clone detection: and (3) selecting a white single colony, inoculating the white single colony in an LB liquid culture medium containing 50mg/LKm, culturing for 4 hours at 250rpm and 37 ℃ with shaking, detecting a positive clone by PCR (polymerase chain reaction), and sequencing and verifying.
3 transformation of Agrobacterium
(1) Taking agrobacterium tumefaciens competent cells stored at the temperature of minus 80 ℃, placing the agrobacterium tumefaciens competent cells on a palm piece, allowing the palm piece to be partially melted, and inserting the palm piece into ice when the palm piece is in an ice-water mixed state;
(2) adding 0.01-1.0 μ g plasmid into 100 μ L competent cells, tapping the tube bottom with finger, mixing, and standing in ice, liquid nitrogen, 37 deg.C water bath and ice bath for 5 min;
(3) adding a sterile LB liquid culture medium, and performing shake culture at 28 ℃ for 2-3 hours;
(4) centrifuging at 6000rpm for 1min to collect bacteria, gently blowing 50 μ L supernatant to obtain heavy suspension bacteria block, spreading on LB plate containing 100mg/L kanamycin, and culturing on 28 deg.C shaking table for 2-3 days
(5) And (3) positive clone detection:
white single colonies were picked, inoculated in LB liquid medium containing 100mg/L kanamycin, cultured with shaking at 250rpm and 37 ℃ for 4 hours, and PCR-detected positive clones were carried out using primers and sent for sequencing verification.
4 infection of callus will be about 1cm in size3Placing the lycoris radiata callus into a liquid culture medium containing transformed agrobacterium tumefaciens MS, infecting for 30min, and continuously shaking during infection, so that the transformed agrobacterium tumefaciens can be favorably invaded into the callus;
and 5, taking out the infected lycoris callus, sucking the infection liquid on the surface of the callus by sterile filter paper, inoculating the callus on a flat plate containing an MS culture medium, and culturing in dark for 3 d. The culture temperature is 25 +/-1 ℃.
6, after the screening culture and the differentiation dark culture of the callus are finished, inoculating the infected callus on a flat plate containing antibiotics. The formula of the culture medium is as follows: 30g/L of sucrose, 7.5g/L of agar, 6.0mg/L of 6-BA, 1.5mg/L of LNAA and 1.0mg/L of multi-wall carbon nano-tube. The types and concentrations of antibiotics were: 250mg/L kanamycin and 200mg/L timentin. The culture temperature is 25 +/-1 ℃, and the culture conditions are as follows: and (5) dark culture. After that, the subculture was performed every 20 d. After callus differentiation, light culture is started with a light intensity of 100 to 300. mu. mol/m-2·s-1And the illumination time is 16 h. Until cluster buds are differentiated.
7, strong bud culture: transferring the bud to a strong bud culture medium for strong bud culture. The formula of the culture medium is as follows: 40g/L of sucrose, 7.5g/L of agar, MS, 2.0mg/L of 6-BA and 0.5mg/L of NAA. Cultivation methodThe cultivation temperature is 25 +/-1 ℃, and the illumination intensity is 100-300 mu mol.m-2·s-1And the illumination time is 16 h. After 30-60 days, the test-tube plantlets can grow into 3-5 test-tube plantlets with root systems of 1-2mm in diameter.
9 rooting culture is carried out by transferring the bud to the strong bud culture medium. After 30 days, the rooting culture is carried out again on the IBA culture medium which is transferred to sucrose 40g/L, agar 7.5g/L, MS, 2.0mg/L6-BA, 0.2mg/LNAA and 2.0 mg/L. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 100-300 mu mol.m-2·s-1And the illumination time is 16 h. After 30 days, the test-tube plantlets can grow into 3-5 test-tube plantlets with root systems of 1-2mm in diameter.
10 identification of transgenic plants
(1) And (3) cutting 0.3cm leaves of the test-tube plantlet, placing the test-tube plantlet in GUS dye solution, and dyeing for more than 12 hours at 37 ℃. Then, absolute ethyl alcohol is used for decoloring. And if the leaves are blue, the reporter gene is successfully integrated into the plant genome, and the plant is a transgenic plant. When the technology is adopted to carry out lycoris transgenic, the dyeing rate of positive plants is 70-80%.
(2) Sequencing and verifying: carrying out PCR verification on the transgenic plant of the blue-dyed plant by adopting a rapid PCR kit produced by Tiangen Biochemical technology (Beijing) Co., Ltd, wherein the used GUS primers are as follows:
upstream primer 5'-GCTGGTCACCAATTCACACG-3' (SEQ ID No.1)
Downstream primer 5'-AGAACACGGGGGACTCTTGA-3' (SEQ ID No.2)
PCR results showed that all blue-stained plants amplified the GUS gene. The PCR system used: MasterMix 10. mu.L, 0.5. mu.L each of the bipartite primers (10. mu.L), 1.0. mu.L of template DNA (20ng/L), and 20. mu.L of double distilled water. PCR program, 94 ℃ 3min, 94 ℃ 30s, 55 ℃ 30s, 721min, 35 cycles, 72 ℃ 5 min. After the PCR was completed, 5. mu.L of the product was collected and subjected to agarose gel electrophoresis. All plants dyed blue can amplify GUS gene with the amplification rate of 100%.
Example 2
Establishment of lycoris radiata anthocyanin synthesis related gene transgenic system
1 Induction of callus Amarylris aseptic bulbils are transferred to sucrose 30g/L + agar 7.5g/L + MS +6.0mg/L6-BA +1.5mg/LNAA medium. After 2 weeks, white protrusions, which are the initiation of callus, appeared around the bulblets, pale yellow tissue appeared around the white protrusions after 20 days, and pale yellow callus clumps appeared after 30 days, at which time subculture was performed again until callus clumps with a diameter of more than 1cm were induced. Culturing at 25 + -1 deg.C in dark.
2 construction of the vector
Sources of genes and vectors: the lycoris radiata anthocyanin synthesis gene MYB119 is synthesized by a gene synthesis company according to a nucleotide sequence shown in SEQ ID No.3 in a sequence table, and enzyme cutting sites SalI and XbaI are added at two ends of the nucleotide sequence. The pCAMBIA2301 plasmid with GUS reporter gene is selected as the carrier. The GUS gene was used as a reporter gene. The sequence of the ligation of the GUS gene and MYB119 on the vector is shown in FIG. 1.
(1) Connecting: MYB119 gene 3. mu.L (20ng/L), cloning vector pCAMBIA 23011. mu.L, T4 ligase, total volume 4. mu.L, reaction at 25 ℃ for 15 min.
(2) And (3) transformation: taking an agrobacterium strain EHA105 stored at the temperature of minus 70 ℃, melting on ice, adding 4 mu L of a connecting product when the strain is in a semi-melting state, flicking and uniformly mixing, carrying out ice bath for 20-30min, carrying out heat shock for 30s at the temperature of 42 ℃, immediately placing on ice for 2min, adding 250 mu L of a nonresistant LB liquid culture medium into an ultra-clean workbench, and carrying out shake culture at the temperature of 37 ℃ for 1 hour at 200 rpm.
(3) Coating a plate: centrifuging, removing supernatant, collecting 100 μ L of bacterial liquid, uniformly spreading on a plate with 50mg/LKm resistance, and culturing at 37 deg.C for 8-12 hr.
(4) And (3) positive clone detection: and (3) selecting a white single colony, inoculating the white single colony in an LB liquid culture medium containing 50mg/LKm, culturing for 4 hours at 250rpm and 37 ℃ with shaking, detecting a positive clone by PCR (polymerase chain reaction), and sequencing and verifying.
3 transformation of Agrobacterium
(1) Taking agrobacterium tumefaciens competent cells stored at the temperature of minus 80 ℃, placing the agrobacterium tumefaciens competent cells on a palm piece, allowing the palm piece to be partially melted, and inserting the palm piece into ice when the palm piece is in an ice-water mixed state;
(2) adding 0.01-1.0 μ g plasmid into 100 μ L competent cells, tapping the tube bottom with finger, mixing, and standing in ice, liquid nitrogen, 37 deg.C water bath and ice bath for 5 min;
(3) adding a sterile LB liquid culture medium, and performing shake culture at 28 ℃ for 2-3 hours;
(4) centrifuging at 6000rpm for 1min to collect bacteria, taking 50 mu L of supernatant, slightly blowing and beating heavy suspension bacteria blocks, coating the heavy suspension bacteria blocks on an LB (Langmuir-Blodgett) flat plate containing 100mg/L kanamycin, and inversely placing the heavy suspension bacteria blocks on a shaking table at 28 ℃ for culturing for 2-3 days
(5) And (3) positive clone detection:
white single colonies were picked, inoculated in LB liquid medium containing 100mg/L kanamycin, cultured with shaking at 250rpm and 37 ℃ for 4 hours, and PCR-detected positive clones were carried out using primers and sent for sequencing verification.
4 infection of callus will be about 1cm in size3Placing the lycoris radiata callus into a liquid culture medium containing transformed agrobacterium tumefaciens MS, infecting for 30min, and continuously shaking during infection, so that the transformed agrobacterium tumefaciens can be favorably invaded into the callus;
and 5, taking out the infected lycoris callus, sucking the infection liquid on the surface of the callus by sterile filter paper, inoculating the callus on a flat plate containing an MS culture medium, and culturing in dark for 3 d. The culture temperature is 25 +/-1 ℃.
6, after the screening culture and the differentiation dark culture of the callus are finished, inoculating the infected callus on a flat plate containing antibiotics. The formula of the culture medium is as follows: 30g/L of sucrose, 7.5g/L of agar, 6.0mg/L of 6-BA, 1.5mg/L of LNAA and 1.0mg/L of multi-wall carbon nano-tube. The types and concentrations of antibiotics were: 250mg/L kanamycin and 200mg/L timentin. The culture temperature is 25 +/-1 ℃, and the culture conditions are as follows: and (5) dark culture. After that, the subculture was performed every 20 d. After callus differentiation, light culture is started with a light intensity of 100 to 300. mu. mol/m-2·s-1And the illumination time is 16 h. Until cluster buds are differentiated.
7, strong bud culture: transferring the bud to a strong bud culture medium for strong bud culture. The formula of the culture medium is as follows: 40g/L of sucrose, 7.5g/L of agar, MS, 2.0mg/L of 6-BA and 0.5mg/L of NAA. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 100-300 mu mol.m-2·s-1And the illumination time is 16 h. After 30-60 days, the test-tube plantlets can grow into 3-5 test-tube plantlets with root systems of 1-2mm in diameter.
9 rooting culture from germination to topAnd (4) on the strong bud culture medium. After 30 days, the rooting culture is carried out again on the IBA culture medium which is transferred to sucrose 40g/L, agar 7.5g/L, MS, 2.0mg/L6-BA, 0.2mg/LNAA and 2.0 mg/L. The culture temperature is 25 +/-1 ℃, and the illumination intensity is 100-300 mu mol.m-2·s-1And the illumination time is 16 h. After 30 days, the test-tube plantlets can grow into 3-5 test-tube plantlets with root systems of 1-2mm in diameter.
10 identification of transgenic plants
(1) And (3) cutting 0.3cm leaves of the test-tube plantlet, placing the test-tube plantlet in GUS dye solution, and dyeing for more than 12 hours at 37 ℃. Then, absolute ethyl alcohol is used for decoloring. And if the leaves are blue, the reporter gene is successfully integrated into the plant genome, and the plant is a transgenic plant. When the technology is adopted for lycoris transgenic, the dyeing rate of positive plants is 60-65%.
(2) Sequencing and verifying: carrying out PCR verification on the transgenic plant of the blue-dyed plant by adopting a rapid PCR kit produced by Tiangen Biochemical technology (Beijing) Co., Ltd, wherein the used GUS gene primers are as follows:
upstream primer 5'-GCTGGTCACCAATTCACACG-3' (SEQ ID No.1)
Downstream primer 5'-AGAACACGGGGGACTCTTGA-3' (SEQ ID No.2)
PCR results showed that all blue-stained plants amplified the GUS gene. The PCR system used: MasterMix 10. mu.L, 0.5. mu.L each of the bipartite primers (10. mu.L), 1.0. mu.L of template DNA (20ng/L), and 20. mu.L of double distilled water. PCR program, 94 ℃ 3min, 94 ℃ 30s, 55 ℃ 30s, 721min, 35 cycles, 72 ℃ 5 min. After the PCR was completed, 5. mu.L of the product was collected and subjected to agarose gel electrophoresis. All plants dyed blue can amplify GUS gene with the amplification rate of 100%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> institute of plant of Chinese academy of sciences of Jiangsu province
Hangzhou vegetable garden (Hangzhou city garden science research institute)
<120> method for constructing lycoris genus genetic transformation system
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gctggtcacc aattcacacg 20
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<213> Artificial Sequence (Artificial Sequence)
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agaacacggg ggactcttga 20
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<213> Artificial Sequence (Artificial Sequence)
<400>3
gtcgacatgg taggctcatt aggagtaagg aaaggtgcat ggacggagga ggaagatata 60
cttctaagga agtgcgttga gaaatatggt gaaggaagat ggcatgaagt tccttccaga 120
gcaggcttga atcgatgcag gaaaagctgc agaatgaggt ggttgaatta tcttaagcca 180
aatgtcaaga gaggacagtt ttcggtggac gaagtggact tgattatcag actacacaag 240
ttgcttggca ataggcaagt gaaaatgtgg tcattgatag ctggtagact ttcaggaaga 300
acagcgaatg atgtaaagaa ttattggaac tcaaaccagc gtaagaaggt gatttctagc 360
actgatgaag ttcaatcaaa accaaaagca aaatcaatca caagagacaa cataataaag 420
cctcaacctt ggaagttcag aaatttattc tggttaagag gaaaaagtac tccacttatt 480
aatgttggtt ctcaatatgg ggacgatctt tgtaagccat gttattcaac agtatcgcca 540
ccttccgaca ttaatgaagt tgaaagtata tggtgggaaa gctcgttaga tgacaaagaa 600
attaatcaaa cgatcaacag cagttgtctg ggttctgttt ctgtttcagc agcagcagct 660
tacctagagt ccagcgaaag tcattttgta aagaacaacg caccaagagg gataaaaact 720
ggggacgtgt tctatgaaca aggacaaaat tgttggagtg acatttcttt ggatgcagac 780
ctttggaatc taatcaatac agaactagat caacaacaac ctgaaggact tcagtctata 840
atgttgtaat ctaga 855
Claims (5)
1. A method for constructing a lycoris genus genetic transformation system is characterized by comprising the following steps:
(1) carrying out enzyme digestion, connection and screening on the exogenous gene and the vector in sequence to obtain a recombinant vector;
(2) introducing the recombinant vector into agrobacterium-mediated cells, carrying out shake culture for 2-3 h, centrifuging, collecting thalli, coating the thalli on a flat plate containing 50-150 mg/L kanamycin resistance, and screening single colonies to obtain positive recombinant agrobacterium;
(3) transferring the positive recombinant agrobacterium to an LB liquid culture medium, performing shake culture at 25-29 ℃ and 180-220 rpm for more than 16h, and culturing to OD of bacterial liquid600Obtaining a positive recombinant agrobacterium liquid when the value reaches 0.4-0.6;
(4) infecting the lycoris callus for 25-35 min by using the positive recombinant agrobacterium liquid to obtain infected lycoris callus;
the preparation method of the lycoris callus comprises the following steps:
s1, transferring the bulblets of the Lycoris to an induction culture medium, and performing dark culture at 24-26 ℃ for 28-32 days to obtain a light yellow callus mass; the induction culture medium consists of an MS culture medium with the following content components: 30g/L of sucrose, 7.5g/L of agar, 6.0mg/L of 6-BA and 1.5 mg/LNAA;
s2, carrying out subculture on the faint yellow callus blocks, and finishing the culture when the diameter of the callus blocks is more than 1 cm;
(5) inoculating the infected lycoris callus on an MS culture medium, and carrying out dark culture for 2-4 d at 24-26 ℃ to obtain dark-cultured lycoris callus;
(6) inoculating the dark cultured lycoris callus on a flat plate containing antibiotics to perform callus differential culture; subculturing every 19-22 days until callus is differentiated to obtain cluster buds;
the antibiotic in the antibiotic-containing plate included the following components at the following concentrations: 200-300 mg/L kanamycin and 150-250 mg/L timentin;
the culture medium in the plate consists of MS culture medium with the following content components: 30g/L of sucrose, 7.5g/L of agar, 6.0mg/L of 6-BA, 1.5mg/L of NAA and 1.0mg/L of multi-wall carbon nano-tube; the pH value of the culture medium is 5.6-5.8;
the illumination intensity of the subculture is 100-300 mu mol.m-2·s-1The illumination time is 8 h;
(7) inoculating the cluster buds to a bud-strengthening culture medium containing antibiotics for bud-strengthening culture, and subculturing once every 19-22 days until bulblets are obtained;
(8) and (4) inoculating the bulblets on a rooting culture medium again for rooting culture to obtain test-tube plantlets with 3-5 roots.
2. The constructing method according to claim 1, wherein the strong bud medium in the step (7) consists of MS medium with the following contents: sucrose 40g/L, agar 7.5g/L, 2.0mg/L6-BA and 0.5 mg/LNAA; the pH value of the strong bud culture medium is 5.6-5.8.
3. The construction method according to claim 1 or 2, wherein the conditions for the strong bud culture in step (7) and the rooting culture in step (8) are independently as follows: the culture temperature is 24-26 ℃, and the illumination intensity is 100-300 mu mol.m-2·s-1And the illumination time is 16 h.
4. The method for constructing a recombinant vector according to claim 1, wherein the exogenous gene in the step (1) comprises a betamine synthesis related gene, an anthocyanin synthesis related gene or a GUS gene.
5. The method for constructing according to claim 4, wherein the recombinant vector comprising the GUS gene in the step (1) is pEF-3-GUS-2; the vector used for transferring the anthocyanin synthesis related gene is pCAMBIA2301, and the multiple cloning sites of the anthocyanin synthesis related gene insertion vector are SalI and XbaI.
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| CN103993037A (en) * | 2014-05-30 | 2014-08-20 | 江苏省中国科学院植物研究所 | Quick transgenic method of lycoris radiata |
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| CN103993037A (en) * | 2014-05-30 | 2014-08-20 | 江苏省中国科学院植物研究所 | Quick transgenic method of lycoris radiata |
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